红景天苷对犬细小病毒体外复制的抑制效应分析

doi:10.11843/j.issn.0366-6964.2024.12.036

摘要/Abstract

摘要：

旨在分析红景天苷(salidroside，SAL)对犬细小病毒(canine parvovirus，CPV)的抑制作用及其机制。对病毒感染过程的3个节点(吸附、侵入、复制)分别添加SAL进行孵育，检测细胞中病毒滴度，评价SAL对病毒的抑制作用；TUNEL (Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling)试验分析细胞凋亡；实时荧光定量PCR和Western blot分别检测细胞凋亡相关蛋白和炎症因子的mRNA或蛋白表达，分析和初步验证SAL抑制病毒复制的机制。结果显示，SAL可显著抑制CPV复制，但对CPV的吸附及侵入过程没有显著影响。SAL可抑制CPV诱导的细胞凋亡，且显著抑制了caspase 8及tBID的蛋白表达。进一步研究发现，CPV可诱导IL-1β及相关炎症因子上调表达，而SAL孵育病毒感染的细胞后部分炎症因子下调表达。Caspase 1、NLRP3的激活与IL-1β密切相关，病毒感染细胞后添加SAL进行孵育可抑制caspase 1的激活，而对NLRP3无显著作用。应用siRNA-caspase 8下调细胞中caspase 8的表达再孵育病毒，结果显示，细胞中IL-1β表达被抑制，与SAL的作用效果一致，表明SAL主要通过抑制caspase 8信号通路活化从而抑制炎症因子的表达。综上所述，SAL可有效抑制CPV的复制，其通过调控caspase 8信号通路抑制了CPV诱导的细胞凋亡，且抑制了部分炎症因子的表达。本研究为有效治疗CPV提供了新的途径和方法。

关键词: 犬细小病毒(CPV), 红景天苷, 细胞凋亡, 细胞因子

Abstract:

This study analyzes the inhibitory effect and its mechanism of salidroside (SAL) on canine parvovirus (CPV). In vitro, drug effects were performed on three stages of virus infection (adsorption, invasion, and replication). Virus titers were detected in cells to access the inhibitory effect of SAL on the virus were evaluated. TUNEL (Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling) assay was used to analyze apoptosis. Quantitative Real-time PCR and Western blot were used to detect the mRNA or protein expression of apoptosis-related proteins and inflammatory factors, respectively, aiming to analyze and preliminarily verify the mechanism by which SAL inhibits virus replication. The results showed that SAL significantly inhibited CPV replication, but had no significant effect on the adsorption and invasion processes of CPV. SAL could inhibit apoptosis induced by CPV and significantly inhibit the protein expression of caspase 8 and tBID. Further research revealed that CPV could induce the upregulation of IL-1β and inflammatory factors, while SAL downregulated the expression of some inflammatory factors in virus-infected cell. The activation of caspase 1 and NLRP3 was closely related to IL-1β. After viral infection of cells, incubation with SAL could inhibit the activation of caspase 1, but had no significant effect on NLRP3. When virus was incubated in cells with down-regulated expression of caspase 8 by siRNA-caspase 8, the expression of IL-1β in cells was inhibited, consistent with the effect of SAL, indicating that SAL mainly inhibits the expression of inflammatory factors by inhibiting the activation of the caspase 8 signaling pathway. In summary, this study shows that SAL can effectively inhibit the replication of CPV. SAL inhibits CPV-induced apoptosis by regulating the caspase 8 signaling pathway and inhibits the expression of some inflammatory factors. This study provides a new approach and method for the effective treatment of CPV.

Key words: canine parvovirus(CPV), salidroside, apoptosis, cytokines

中图分类号:

S852.659.2

张美雯, 王成龙, 刘玉贞, 赵育桐, 朱记平, 李毅. 红景天苷对犬细小病毒体外复制的抑制效应分析[J]. 畜牧兽医学报, 2024, 55(12): 5738-5750.

ZHANG Meiwen, WANG Chenglong, LIU Yuzhen, ZHAO Yutong, ZHU Jiping, LI Yi. Analysis of the Inhibitory Effect of Salidroside on Canine Parvovirus Replication in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5738-5750.

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基因 Gene	正向引物(5′→3′) Forword primer(5′→3′)	反向引物(5′→3′) Reverse primer(5′→3′)
Caspase 8	ACAAGGGCATCATCTATGGCTCTGA	CCAGTGAAGTAAGAGGTCAGCTCAT
Caspase 12	GCCGTCTGGGTGACTGATG	CTGCAAGGGCTGGTCACAT
p53	TAACAGTTCCTGCATGGGCGGC	AGGACAGGC ACAAACACGCACC
Bax	TTCCGAGTGGCAGCTGAGATGTTT	TGCTGGCAAAGTAGAAGAGGGCAA
Bcl2	CATGCCAAGAGGGAAACACCAGAAG	GTGCTTTGCATTCTTGGATGAGGG
FasL	GAGGAGGGACCACAACACAGGTCTC	AGAGCTGAAACATCCCCAGC
Fas	ACTGTGCAGACATCGACCTG	CGTTGATCCCGTTCTTCCGA
FADD	TGGAGGAGACTGGCTCGTTA	CTTGTTGGCTGGCTCCAAAC
IL-8	TGGAAATGAGGTGTGCCTGG	GGATCTTGTTTCTCAGCCTTCTT
IL-7	CAGCATCAATGACTTGGACATCA	TTCAACTTGTGAGCAGCACG
IL-6	CTCCTGGTGATGGCTACTGC	GTGCAGAGATTTTGCCGAGG
TNF-α	GTGCCGTCAGATGGGTTGTA	CAAGGGCTCTTGATGGCAGA
CCL5	GACTACCCTTCACCCACGTC	CTTTCGGGTGACAAAGACGAC
CXCL10	TGACTCTGAATGGTACTCAAGGA	GACACGATGGACTTGCAGGA
IFNB	GCGAGAAATCACGCCAGTTC	TCTATTGTCCAGGCACAGATGC
GAPDH	GCTGCCCAGAACATCATCC	GTCAGATCCACGACGGACAC

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